Throughout this disclosure reference will be made to the published literature by numerals in parenthesis. These numerical references correspond to a listing of such literature references appearing at the end of this disclosure; all of these literature references being hereby incorporated herein by reference.
Viruses infect cells and divert the biosynthetic apparatus of the cell to synthesizing virus progeny. Certain viruses infect the host cell, cause the host DNA to break down and virus progeny to form in the cell whereupon the cell lyses with the release of mature virus progeny. Other viruses are lysogenic. These viruses infect a host cell and the viral DNA becomes inserted into a region of the host chromosome whereupon for generations the resultant cell-line (from replication of the infected cell) expresses genetic products of the virus. A progeny of the original infected cell can spontaneously release the viral DNA from its chromosome or be induced to do the same, whereupon a lytic cycle resulting in viral progeny occurs. An example of this latter type of virus is the phage lambda which on certain occasions, e.g., exposure to certain chemicals or radiation such as ultraviolet light, may initiate a lytic cycle immediately after infection but can otherwise exist as a provirus or prophage in the E. coli host genome for many generations.
A recent development in the field of antibodies is the amplification by the polymerase chain reaction (PCR) of nucleotide sequences for antibodies or portions thereof (1). An extension of this development is the insertion of these sequences into the genome of viruses, especially phages or bacteriophages (2, 3, 4, 11). In this regard reference is expressly made to PCT Patent Publication WO920 1047, published Jan. 23, 1992 entitled "Methods For Producing Members of Specific Binding Pairs," incorporated herein by reference. Likewise, the expression of a catalytically-active enzyme on the surface of a phage has been achieved (32).
For instance, Clackson et al. (2) report using a random combinational library of rearranged sequences for heavy (V.sub.h) and kappa (V.sub.k) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx) to display diverse libraries of antibody fragments on the surface of the fd phage. The recombinant fd phages were selected by passing the population thereof over an affinity column.
Likewise, McCafferty et al. (3) report that complete antibody V domains can be displayed on the surface of a recombinant fd bacteriophage and that those that bind to an antigen (e.g., one in a million) can be isolated by affinity chromatography. And, McCafferty et al. (32) report the expression and affinity chromatography of functional alkaline phosphatase on the surface of a bacteriophage.
Similarly, Huse et al. (4) relate employing the bacteriophage lambda vector system to express in E. coli a combinatorial library of Fab fragments. Selection for expression was by selection for binding to an antigen.
A problem with the technique of selection of recombinant phages suspected of expressing catalytic antibodies or catalytically active portions thereof by hapten or antigen binding or affinity is that initially an enormous number of phages are produced; for instance, of the order of greater than 10.sup.5. Selection for hapten-binding from this enormous population of phages still yields an enormous subpopulation of phages (that bind); for instance, of the order of 6,000-10,000 phages. However, in this first subpopulation that bind there is yet a smaller second subpopulation that not only express the antibody on their surface (and therefore bind to the hapten), but, also display a catalytic antibody (i.e., the antibody or portion thereof expressed is catalytic). Thus, isolation of only the first subpopulation (that bind with the antigen or hapten) does not adequately screen the recombinant phage population to isolate those members which express the antibody or portion thereof catalytically. That is, hapten-binding selection is insufficient: to isolate those members of the recombinant phage population which express catalytic antibodies or portions thereof for further use; e.g., for infecting a host cell such as E. coli and producing consistent generations of recombinant phage or cells expressing the catalytic antibody or a catalytic portion thereof. Indeed, in a broader scope, a problem facing the development of catalysts such as catalytic antibodies, is the inability to economically enrich or select for moieties, e.g., antibodies, exhibiting the desired catalytic activity from among a vast excess of non-catalytic moieties, e.g., a vast excess of non-catalytic antibodies raised against the same transition state analogs.
Further, prior methods for selection of catalytic activity of antibody fragments (as opposed to their identification through extensive selection exercises) depends on biological selection based on the ability to compliment genetic defect in an organism expressing the fragment (16).
Heretofore there has been no method for selection of recombinant viruses or cells infected by such viruses displaying catalytic antibodies or catalytic portions thereof based upon catalytic properties of such viruses or cells.
In the area of enzymology the literature (5, 6) reports reactants called mechanism-based inhibitors (affinity labels or suicide substrates). These reactants bind in the active site of an enzyme as normal substrates do, but, contrary to normal substrates, exploit the chemical features of the reaction mechanism to form an irreversible adduct with the enzyme. Such reactants have been specifically designed for many enzymes and enzyme mechanisms. Generally, a nucleophilic enzyme amino acid residue that participates in the normal substrate catalytic reaction reacts instead with the mechanism-based inhibitor and is permanently inactivated. Haptens which were suicide substrates have been used to elicit antibodies (14). The suicide substrates were not used for selection of antibodies having catalytic activity.
Thus, heretofore there has been no application of mechanism-based inhibitors to select recombinant phage or recombinant phage infected cell populations for members expressing a catalytic antibody or catalytic portion thereof or to increase the concentration of members expressing catalytic moieties. Nor has there been any application of mechanism-based inhibitors to screen for catalytic moieties, such as catalytic moieties expressed by phages, cells, or other self-replicating systems, or catalytic peptides, oligopeptides, polypeptides, or enzymes. Nor has there been any application of mechanism-based inhibitors to increase the concentration of catalytic moieties in a sample containing catalytic moieties.
Work with enzymes show that active enzymes can "crawl" across a two-dimensional surface covered with substrate (on a micrometer distance scale), while inactive enzymes with the same binding affinity for the substrate are greatly restricted in their mobility (7, 8, 9). However, heretofore there has been no application of a two dimensional surface including a substrate of a desired catalytic reaction for selection of recombinant phage or recombinant phage infected cell population for members expressing a catalytic antibody or catalytic portion thereof or to increase the concentration of members expressing catalytic moieties. Nor has there been any application of catalysis-accelerated movement to select for catalytic moieties, or to increase the concentration of catalytic moieties in a sample containing catalytic moieties.
The kinetics of antibody binding to solid-phase immobilized antigen have been investigated (10). However, non-catalytic moieties have not been separated from catalytic moieties on the basis of surface binding. For instance, recombinant phages or recombinant phage infected cells have not been screened or concentrated on the basis of those which express a non-catalytic antibody binding to the substrate with the same affinity, regardless of incubation time, whereas those which express a catalytic antibody or catalytic portion thereof initially binding to the substrate, but dissociating once catalysis has occurred. Nor has surface binding been employed to select for catalytic moieties in a sample containing catalytic moieties.
In addition, while effects of temperature on binding and catalysis by enzymes has been investigated (12), heretofore there has been no use of the discontinuity in the substrate binding of a catalytic moiety (but not of a non-catalyst) as a function of temperature to select for catalytic moieties, increase the concentration of catalytic moieties in a sample containing catalytic moieties or to screen or concentrate recombinant phages or recombinant phage infected cells expressing catalytic moieties.
While principles of "weak affinity chromatography" in the presence of a variety of competing soluble ligands to alter the retention of molecules on chromatographic columns, for instance of a ligand which possesses relatively weak affinity for a moiety covalently coupled to a solid support (17) and catalytic mechanisms (15) have been examined, heretofore there has been no application of changes in binding of catalysts by competition to isolate or select catalysts from non-catalysts or to increase the concentration of catalysts.
It is desired to be able to select for catalytic moieties on the basis of those kinetic and thermodynamic properties intrinsically and essentially associated with catalysis, i.e., reaction-based selection is desired. It is also desired to be able to increase the concentration of catalytic moieties in a sample, and to obtain this increased concentration by exploiting catalytic properties, i.e., obtaining an increased concentration of catalytic moieties on a reaction basis. It is further desired to screen or concentrate a recombinant virus or recombinant virus infected cell population which expresses a catalytic moiety on the basis of catalytic properties, i.e., reaction based selection or concentrating of such a population.
As mentioned earlier, it is also desired to be able to screen a recombinant phage population not only for those members expressing an antibody (e.g., by affinity or hapten binding), but, to also screen this population for members which express a catalytic antibody or portion thereof, a catalytic enzyme, or more generally a catalytic moiety. For instance, selection based on catalytic properties is desired so that those members of the population which so express the catalytic antibody or portion thereof can be used to produce further populations (without substantial contamination by members that do not express the antibody or portion thereof or that express it but not catalytically), or to catalyze desired reactions (with optimal turnover rate due to minimal contamination by or reduced concentration of members that do not express the catalytic antibody or portion thereof). Indeed, in the scenario of attempting to use recombinant phages or the products of recombinant phage infected cells to catalyze a reaction, those members of the population that express the antibody or portion thereof, but not in a catalytically active form, are deleterious to the reaction system because they can compete with catalytic phages or moieties for substrate. Likewise, with respect to using a recombinant phage population to infect cells and produce monoclonal antibodies, reaction based selection of the population is desired to reduce the labor involved in otherwise reducing the population to a smaller population to further create monoclonal antibodies. Thus, it is desired to be able to perform reaction based selection or concentrating of a recombinant phage or recombinant phage infected cell population for catalytic activity.